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RHAMM deficiency disrupts folliculogenesis resulting in female hypofertility.

Li H, Moll J, Winkler A, Frappart L, Brunet S, Hamann J, Kroll T, Verlhac MH, Heuer H, Herrlich P, Ploubidou A - Biol Open (2015)

Bottom Line: This resulted in folliculogenesis defects and female hypofertility, although HA-induced signalling was not affected.Deletion of the RHAMM C-terminus in vivo abolishes its spindle association, resulting in impaired spindle orientation in the dividing granulosa cells, folliculogenesis defects and subsequent female hypofertility.These data reveal the first identified physiological function for RHAMM, during oogenesis, and the importance of this spindle-associated function for female fertility.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Institute for Age Research - Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany.

No MeSH data available.


Deletion of the RHAMM C-terminus results in expression of a truncated protein variant and consequent female hypofertility, but it does not prevent HA-induced signalling.(A) Schematic representation of the strategy employed in the generation of the hmmrm/m mouse. A neomycin-resistance cassette (neo) with an in-frame stop codon was targeted into the genomic sequence of wild type hmmr+/+, replacing part of exons 10 and 11 as well as the intronic region. (B) The resulting premature translation stop gave rise to a truncated (RHAMM ΔC) protein, lacking the HA-binding and centrosome-targeting domain of wild type RHAMM, and fused to the neomycin-resistance gene product neomycin phosphotransferase (NPT). (C) RT-PCR analysis, employing primer pairs targeting different RHAMM genomic regions, demonstrates the insertion of the neomycin cassette, as indicated by the increased size of the PCR gene product in the hmmrm/m sample, in the region of exons 7–12. GAPDH was used as control. (D) The full length (95 kDa) RHAMM protein is expressed in hmmr+/+ MEFs, while their mutated hmmrm/m counterparts express a truncated 65 kDa protein (asterisk) fused to the NPT gene product, as demonstrated by the western blot probed with anti-RHAMM and anti-NPT antibodies. α-tubulin was used as loading control. (E) Deletion of the RHAMM C-terminus does not prevent HA-induced ERK1/2 activation, as indicated by the level of phosphorylated ERK1/2 (p-ERK1/2) in hmmrm/m MEFs compared to wild type hmmr+/+ controls, assessed by western blotting. ERK1/2 and α-tubulin were used as loading controls. (F,G) Average number of litters (F) and litter size (G) (denoted by the average number of offspring per litter), produced by mating pairs of the indicated genotype over a period of 6 months. The mice used in the breeding assay were 8–12 weeks old. (H) Age-dependent female hypofertility, induced by the RHAMM deficiency, is indicated by the reduction in litter size of hmmrm/m females older than 24 weeks. A minimum of 25 litters per genotype and time point was quantified. In F,G,H, data are presented as mean±s.d.; n indicates the number of mating pairs per group, *p<0.05.
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f02: Deletion of the RHAMM C-terminus results in expression of a truncated protein variant and consequent female hypofertility, but it does not prevent HA-induced signalling.(A) Schematic representation of the strategy employed in the generation of the hmmrm/m mouse. A neomycin-resistance cassette (neo) with an in-frame stop codon was targeted into the genomic sequence of wild type hmmr+/+, replacing part of exons 10 and 11 as well as the intronic region. (B) The resulting premature translation stop gave rise to a truncated (RHAMM ΔC) protein, lacking the HA-binding and centrosome-targeting domain of wild type RHAMM, and fused to the neomycin-resistance gene product neomycin phosphotransferase (NPT). (C) RT-PCR analysis, employing primer pairs targeting different RHAMM genomic regions, demonstrates the insertion of the neomycin cassette, as indicated by the increased size of the PCR gene product in the hmmrm/m sample, in the region of exons 7–12. GAPDH was used as control. (D) The full length (95 kDa) RHAMM protein is expressed in hmmr+/+ MEFs, while their mutated hmmrm/m counterparts express a truncated 65 kDa protein (asterisk) fused to the NPT gene product, as demonstrated by the western blot probed with anti-RHAMM and anti-NPT antibodies. α-tubulin was used as loading control. (E) Deletion of the RHAMM C-terminus does not prevent HA-induced ERK1/2 activation, as indicated by the level of phosphorylated ERK1/2 (p-ERK1/2) in hmmrm/m MEFs compared to wild type hmmr+/+ controls, assessed by western blotting. ERK1/2 and α-tubulin were used as loading controls. (F,G) Average number of litters (F) and litter size (G) (denoted by the average number of offspring per litter), produced by mating pairs of the indicated genotype over a period of 6 months. The mice used in the breeding assay were 8–12 weeks old. (H) Age-dependent female hypofertility, induced by the RHAMM deficiency, is indicated by the reduction in litter size of hmmrm/m females older than 24 weeks. A minimum of 25 litters per genotype and time point was quantified. In F,G,H, data are presented as mean±s.d.; n indicates the number of mating pairs per group, *p<0.05.

Mentions: The functions of RHAMM, thus far described in cultured cells, are mediated by its centrosome-targeting and HA-binding domains, which are located in the C-terminus (Fig. 2B) and are responsible for the spindle assembly and cell migration roles of the protein, respectively. To inactivate this region in the mouse, a neomycin cassette with in-frame stop codons was inserted in the mouse genome, replacing a region between exon 10 and 11 of hmmr (Fig. 2A).


RHAMM deficiency disrupts folliculogenesis resulting in female hypofertility.

Li H, Moll J, Winkler A, Frappart L, Brunet S, Hamann J, Kroll T, Verlhac MH, Heuer H, Herrlich P, Ploubidou A - Biol Open (2015)

Deletion of the RHAMM C-terminus results in expression of a truncated protein variant and consequent female hypofertility, but it does not prevent HA-induced signalling.(A) Schematic representation of the strategy employed in the generation of the hmmrm/m mouse. A neomycin-resistance cassette (neo) with an in-frame stop codon was targeted into the genomic sequence of wild type hmmr+/+, replacing part of exons 10 and 11 as well as the intronic region. (B) The resulting premature translation stop gave rise to a truncated (RHAMM ΔC) protein, lacking the HA-binding and centrosome-targeting domain of wild type RHAMM, and fused to the neomycin-resistance gene product neomycin phosphotransferase (NPT). (C) RT-PCR analysis, employing primer pairs targeting different RHAMM genomic regions, demonstrates the insertion of the neomycin cassette, as indicated by the increased size of the PCR gene product in the hmmrm/m sample, in the region of exons 7–12. GAPDH was used as control. (D) The full length (95 kDa) RHAMM protein is expressed in hmmr+/+ MEFs, while their mutated hmmrm/m counterparts express a truncated 65 kDa protein (asterisk) fused to the NPT gene product, as demonstrated by the western blot probed with anti-RHAMM and anti-NPT antibodies. α-tubulin was used as loading control. (E) Deletion of the RHAMM C-terminus does not prevent HA-induced ERK1/2 activation, as indicated by the level of phosphorylated ERK1/2 (p-ERK1/2) in hmmrm/m MEFs compared to wild type hmmr+/+ controls, assessed by western blotting. ERK1/2 and α-tubulin were used as loading controls. (F,G) Average number of litters (F) and litter size (G) (denoted by the average number of offspring per litter), produced by mating pairs of the indicated genotype over a period of 6 months. The mice used in the breeding assay were 8–12 weeks old. (H) Age-dependent female hypofertility, induced by the RHAMM deficiency, is indicated by the reduction in litter size of hmmrm/m females older than 24 weeks. A minimum of 25 litters per genotype and time point was quantified. In F,G,H, data are presented as mean±s.d.; n indicates the number of mating pairs per group, *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400598&req=5

f02: Deletion of the RHAMM C-terminus results in expression of a truncated protein variant and consequent female hypofertility, but it does not prevent HA-induced signalling.(A) Schematic representation of the strategy employed in the generation of the hmmrm/m mouse. A neomycin-resistance cassette (neo) with an in-frame stop codon was targeted into the genomic sequence of wild type hmmr+/+, replacing part of exons 10 and 11 as well as the intronic region. (B) The resulting premature translation stop gave rise to a truncated (RHAMM ΔC) protein, lacking the HA-binding and centrosome-targeting domain of wild type RHAMM, and fused to the neomycin-resistance gene product neomycin phosphotransferase (NPT). (C) RT-PCR analysis, employing primer pairs targeting different RHAMM genomic regions, demonstrates the insertion of the neomycin cassette, as indicated by the increased size of the PCR gene product in the hmmrm/m sample, in the region of exons 7–12. GAPDH was used as control. (D) The full length (95 kDa) RHAMM protein is expressed in hmmr+/+ MEFs, while their mutated hmmrm/m counterparts express a truncated 65 kDa protein (asterisk) fused to the NPT gene product, as demonstrated by the western blot probed with anti-RHAMM and anti-NPT antibodies. α-tubulin was used as loading control. (E) Deletion of the RHAMM C-terminus does not prevent HA-induced ERK1/2 activation, as indicated by the level of phosphorylated ERK1/2 (p-ERK1/2) in hmmrm/m MEFs compared to wild type hmmr+/+ controls, assessed by western blotting. ERK1/2 and α-tubulin were used as loading controls. (F,G) Average number of litters (F) and litter size (G) (denoted by the average number of offspring per litter), produced by mating pairs of the indicated genotype over a period of 6 months. The mice used in the breeding assay were 8–12 weeks old. (H) Age-dependent female hypofertility, induced by the RHAMM deficiency, is indicated by the reduction in litter size of hmmrm/m females older than 24 weeks. A minimum of 25 litters per genotype and time point was quantified. In F,G,H, data are presented as mean±s.d.; n indicates the number of mating pairs per group, *p<0.05.
Mentions: The functions of RHAMM, thus far described in cultured cells, are mediated by its centrosome-targeting and HA-binding domains, which are located in the C-terminus (Fig. 2B) and are responsible for the spindle assembly and cell migration roles of the protein, respectively. To inactivate this region in the mouse, a neomycin cassette with in-frame stop codons was inserted in the mouse genome, replacing a region between exon 10 and 11 of hmmr (Fig. 2A).

Bottom Line: This resulted in folliculogenesis defects and female hypofertility, although HA-induced signalling was not affected.Deletion of the RHAMM C-terminus in vivo abolishes its spindle association, resulting in impaired spindle orientation in the dividing granulosa cells, folliculogenesis defects and subsequent female hypofertility.These data reveal the first identified physiological function for RHAMM, during oogenesis, and the importance of this spindle-associated function for female fertility.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Institute for Age Research - Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany.

No MeSH data available.